After the blood coagulation process has been initiated, the coagulation cascade continues through sequential activation of various proenzymes (zymogens) in the blood to their active forms, the serine proteases. Among them are, inter alia, Factor XII/XIIa, Factor XI/XIa, Factor IX/IXa, Factor X/Xa, Factor VII/VIIa and prothrombin/thrombin. In their physiological state, most of these enzymes are only active if associated to a membrane surface in a complex. Ca ions are involved in many of these processes. The blood coagulation will either follow the intrinsic pathway, wherein all protein components are present in the blood, or the extrinsic pathway, wherein the cell membrane tissue factor plays a critical role. Finally, the wound will be closed by thrombin cleaving fibrinogen to fibrin.
The prothrombinase complex is responsible for activating prothrombin to thrombin. Thrombin is an important enzyme which can act as a procoagulant as well as an anticoagulant. The prothrombinase complex, in which, inter alia, Factor Va (as cofactor) and Factor Xa (as serine protease) are involved, assembles in a Ca-dependent association at the surface of phospholipids. It is discussed that Factor Xa is the catalytic component of the prothrombinase complex.
Factor X (Stuart-Prower factor) is a vitamin K-dependent coagulation glycoprotein by which the intrinsic and the extrinsic blood coagulation cascades can be activated. The primary translation product of Factor X (pre-pro-FX) has 488 amino acids and is initially synthesized by the liver or human hepatoma cells as a single-chain 75 kD precursor protein. In plasma, Factor X is largely present as a double chain molecule (Fair et al., 1984, Blood 64:194–204).
During biosynthesis, after cleavage of the presequence by a signal peptidase (between Ser23/Leu24) and of the propeptide (between Arg40/Ala41), the single chain Factor X molecule is cleaved by processing and removal of the tripeptide Arg180-Lys181-Arg182 to the double chain form consisting of the approximately 22 kD light chain and the approximately 50 kD heavy chain, which are connected via a disulfide bridge (FIGS. 1A–1B). Therefore, Factor X circulates in the plasma as a double chain molecule.
During the blood coagulation process, Factor X is converted from inactive zymogen to active protease Factor Xa by limited proteolysis; wherein Factor X can be activated to Factor Xa in either of two membrane-associated complexes: in the extrinsic Factor VIIa-tissue factor complex or in the intrinsic Factor VIIIa-Factor IXa-phospholipid-Ca-complex, or “tenase complex” (Mertens et al., 1980, Biochem. J. 185:647–658). A proteolytic cleavage between amino acids Arg234/Ile235 results in the release of an activation peptide having a length of 52 amino acids from the N-terminus of the heavy chain and thus to the formation of the active enzyme, Factor Xa. The catalytic center of Factor Xa is located on the heavy chain.
Activation via the Factor VIIa-TF (extrinsic) complex results in the formation of Factor Xaα (35 kD) and Factor Xaβ (31 kD), with a polypeptide of 42 (kD) forming, too, if the Factor VIIa concentration in the complex is low. Factor Xaα is formed by a cleavage at Arg234/Ile235 of the heavy chain and represents the activation of Factor X to Factor Xa. The occurence of Factor Xaβ presumably results from an autocatalytic cleavage at Arg469/Gly470 in the C-terminus of the heavy chain of Factor Xaα and the removal of a 4.5 kD peptide. Like Factor Xaα, Factor Xaβ has catalytic activity. It has been shown, however, that a plasminogen binding site is formed by the cleavage of Factor Xaα to Factor Xaβ, and that Factor Xaβ optionally has fibrinolytic activity or is involved in fibrinolysis as a cofactor. The conversion of Factor Xα to Factor Xaβ, however, is slower than the formation of thrombin, thus preventing the initiation of fibrinolysis before a blood clot is formed (Pryzdial et al., 1996, J. Biol. Chem. 271:16614–16620; Pryzdial et al., 1996, J. Biol. Chem. 271:16621–16626).
The 42 kD polypeptide results from processing in the C-terminus of the heavy chain between Arg469/Gly470 without previous processing between Arg234/Ile235. Like a Factor Xaγ fragment formed by proteolysis at Lys370, this intermediate has no catalytic activity (Mertens et al., 1980, Biochem. J. 185:647–658; Pryzdial et al., 1996, J. Biol. Chem. 271:16614–16620).
Intrinsic Factor X activation is catalysed by the Factor IXa-Factor VIIIa complex. The same processing products are obtained during activation, but the Factor Xaβ product is obtained in larger quantity than other Factor X processing products (Jesty et al., 1974, J. Biol. Chem. 249:5614).
In vitro, Factor X can, for instance, be activated by Russell's viper venom (RVV) or trypsin (Bajaj et al., 1973, J. Biol. Chem. 248:7729–7741) or by purified physiological activators, such as FVIIa-TF complex or Factor IXa-Factor VIIIa complex (Mertens et al., 1980, Biochem. J. 185:647–658).
Most commercially available Factor X products from plasma contain a mixture of Factor Xα and Factor Xaβ, because after activation of Factor X to Factor Xa mainly Factor Xaα is formed, which is, in turn, cleaved to Factor Xaβ in an autocatalytic process. In order to produce a uniform Factor Xa product having high structural integrity, EP 0 651 054 suggested to activate Factor X with RVV over an extended period of time so that the resulting final product substantially contains Factor Xaβ. The by-products, e.g. Factor Xaα, as well as the protease were subsequently removed by several chromatographic steps.
Factor X cDNA has been isolated and characterized (Leytus et al., 1984, Proc. Natl. Acad. Sci., U.S.A., 82:3699–3702; Fung et al., 1985, Proc. Natl. Acad. Sci., U.S.A., 82:3591–3595). Human Factor X has been expressed in vitro in various types of cells, such as human embryonal kidney cells or CHO cells (Rudolph et al., 1997, Prot. Expr. Purif. 10:373–378; Wolf et al., 1991, J. Biol. Chem. 266:13726–13730). However, it has been found that in the recombinant expression of human Factor X, the processing at position Arg40/Ala41 is inefficient, as opposed to the situation in vivo, and that different N-termini at the light chain of Factor X are produced (Wolf et al., 1991, J. Biol. Chem. 266:13726–13730). Recombinant Factor X (rFX) was activated to rFactor Xa (rFXa) by RVV in vitro, or rFXa was expressed directly, with the activating peptide being deleted from amino acid 183 to amino acid 234 and replaced by a tripeptide in order to allow processing directly to a double chain rFXa form. About 70% of purified rFX was processed into light and heavy chain, while the remaining 30% represented single chain rFX of 75 kD. Direct expression of rFXa did result in the formation of active Factor Xa, but also of inactive intermediates. Wolf et al. (1991, J. Biol. Chem. 266:13726–13730) detected still reduced activity of recombinant Factor X, which they ascribed to the poorer ability of rFX to be activated by RVV and to the inactive protein and polypeptide populations of the single chain precursor molecule. In particular, they found high rFXa instability when expressed by recombinant cells, which they ascribed to the high rate of autoproteolysis.
In order to study the function of the C-terminal peptide of Factor Xaα, Eby et al. (1992, Blood 80 (Suppl. 1):1214 A) introduced a stop codon at position Gly430 of the Factor X sequence. However, they did not find a difference between the rate of activation of Factor Xa (FXaα) by β-peptide or a deletion mutant without β-peptide (FXaβ).
Factor Xa is an important component of the prothrombinase complex and might therefore be used to treat patients suffering from blood coagulation disorders, e.g. hemophilia.
Particularly the treatment of hemophilia patients suffering from Factor VIII or Factor IX deficiency with factor concentrates produced from plasma is often complicated by the formation of inhibiting antibodies against these factors in long-term therapy. Therefore, a number of alternatives have been developed to treat hemophiliacs with factors having bypass activity. The use of prothrombin complex concentrate, partially activated prothrombinase complex (APPC), Factor VIIa or FEIBA has been suggested. Commercial preparations with Factor VIII bypass activity (FEIBA) are, for instance, FEIBA® or Autoplex®. FEIBA® contains comparable units of Factor II, Factor VII, Factor IX, Factor X and FEIBA, small amounts of Factor VIII and Factor V, and traces of activated coagulation factors, such as thrombin and Factor Xa or a factor having Factor X-like activity (Elsinger, 1982, Activated Prothrombin Complex Concentrates. Ed. Mariani, Russo, Mandelli, pp. 77–87). Elsinger particularly points at the importance of a “Factor Xa-like” activity in FEIBA®. Factor VIII bypass activity was shown by Giles et al (1988, British J. Haematology 9:491–497) for a combination of purified Factor Xa and phospholipids in an animal model.
Therefore, Factor X/Xa or Factor X/Xa-like proteins, either alone or as a component of a coagulation complex, are in high demand and can be used in various fields of application in hemostasis therapy.
In vivo as well as in vitro, the half-life of Factor Xa is considerably shorter than the half-life of the zymogen. For instance, Factor X can be stored stably in glycerol for 18 months, while Factor Xa is stable for only 5 months under the same conditions (Bajaj et al., 1973, J. Biol. Chem. 248:7729–7741) and shows reduced activity by more than 60% after 8 months in glycerol at 4° C. (Teng et al., 1981, Thrombosis Res. 22:213–220). The half-life of Factor Xa in serum is a mere 30 seconds.
Because Factor Xa is instable, the administration of Factor X preparations has been suggested (U.S. Pat. No. 4,501,731). If, however, the bleeding is-so serious that the patient might die, particularly in hemophiliacs, the administration of Factor X is ineffective, because owing to the functional “tenase complex” deficiency in the intrinsic pathway of blood coagulation, Factor X can not be sufficiently activated to Factor Xa, and activation via the extrinsic pathway is often too slow to show effects quickly. Moreover, hemophiliacs have sufficient amounts of Factor X, but its prothrombinase activity is 1000 times less than that of Factor Xa. In such cases it is necessary to administer activated Factor Xa directly, optionally in combination with phospholipids, as described in Giles et al. (1988, British J. Haematology 9:491–497) or with other coagulation factors, e.g. with Factor VIII bypass activity.
In the preparation of Factor Xa from Factor X, activation has so far mostly been carried out by nonphysiological activators of animal origin, such as RVV or trypsin, and it was necessary to make absolutely sure that the final product is completely free of these proteases. As mentioned above, when Factor X is activated to Factor Xa, quite a number of intermediates, some of them inactive, are formed (Bajaj et al., 1973, J. Bio. Chem. 248:7729–7741; Mertens et al., 1980, Biochem. J. 185:647–658). The presence of such intermediates results in reduced specific activity of the product and may produce intermediates which can function as active serine protease antagonists. Therefore, the preparation of a uniform, pure product having high specific activity according to conventional methods requires complex processes of activation and chromatographic purification.